It is well-known that certain enzymes convert aqueous aldose monosaccharides to ketose monosaccharides and vice versa. Within recent years, isomerases have attracted considerable commercial interest in glucose syrup isomerization reactions. Such enzymes are frequently referred to by the trade as glucose isomerases. Most isomerases can isomerize a plurality of aldose and/or ketose monosaccharide substrates. For example, many glucose isomerases can also isomerize xylose.
Fully hydrated glucose isomerases will deactivate upon storage. Deactivation is accelerated at elevated temperatures. An isomerase manufacturer will, therefore, customarily retail it as a dry isomerase. In order to use this dry isomerase in an isomerization process, the isomerized syrup manufacturer must convert it to the hydrated form.
A typical commercial glucose isomerization process results in an isomerized glucose syrup product containing approximately 45-55 parts fructose and 55-45 parts glucose. The glucose isomerization process normally requires from about 24 to 72 hours and will be conducted at temperatures above 50.degree. C. (usually between 60.degree. to 70.degree. C.). Achieving and maintaining sufficient isomerase activity throughout the isomerization process is necessary in order to obtain the desired high fructose yield.
Unfortunately, isomerases are inherently susceptible to deactivation especially when employed for prolonged periods at the elevated isomerization media temperature as required to achieve optimum dextrose isomerization. In batch processes, reduced isomerase activity is normally compensated by charging the reactor with a sufficiently large enough isomerase dosage to permit the isomerization reaction to proceed to completion. In a continuous isomerization process, isomerase deactivation may be partially corrected by periodic addition of fresh isomerase to the isomerization reactor. This isomerase deactivation problem creates difficulties and additional expense in the manufacture of isomerized glucose syrups. Isomerases are expensive and, therefore, any significant decrease in isomerase requirements would result in substantial cost reductions to the isomerized glucose syrup manufacturer. Also, an isomerase excess can readily lead to the formation of undesirable by-products which, unless removed therefrom, will reduce the quality and value of the isomerized syrup product. Removal of these by-product impurities necessitates additional processing steps, frequently lowers production capacity and results in increased capital equipment expenditures which adversely affects overall production costs.
Continuous fixed isomerase bed reactors are employed in producing a substantial percentage of the isomerized glucose syrups today. In continuous fixed bed reactor systems, the desired fructose level is obtained by permitting a high glucose containing syrup to flow through a bed of immobilized glucose isomerase or a series of reactor beds. Passage of the glucose containing syrup through the fixed isomerase beds is discontinued upon achieving the desired fructose conversion. The amount of fructose produced by a fixed bed reactor is directly proportional to its isomerase activity. Decreased yields inherently arise as a result of isomerase deactivation therein. The isomerase bed will ultimately deteriorate and become totally ineffective. Removal of the deactivated isomerase from the fixed bed or partial replenishment with fresh isomerase is necessary to insure continued fructose production. Periodic or frequent replacement of fresh isomerase substantially reduces overall isomerized glucose syrup capacity. To compensate for these problems, the isomerized glucose syrup producer may install relatively large fixed bed reactors or additional fixed bed reactors (an added capital expenditure), increase the number of passes of the glucose syrup through the bed, reduce glucose syrup flow rate through the fixed bed reactor and thereby increase the contact time therewith, utilize excessive amounts of isomerase in the beds (undesirable because it usually reduces bed flow rates and concomitant production of undesirable by-products) and such other corrective processing modifications.
Several alternatives have been proposed to overcome this isomerase deactivation problem. Many researchers deem the solution to isomerization deactivation problem as residing in the discovery of an organism which will produce a more stable form of isomerase. As a result, numerous existing organisms and new strains have been screened and isolated in the art's attempt to discover a more stable isomerase.
Immobilized isomerases have also been proposed as a means for inhibiting isomerase deactivation since free or water-soluble isomerases are relatively unstable. Heat or chemical treatment of viable cells containing intracellular isomerase, encapsulation, complexing of the isomerase with natural and synthetic polymers, immobilization of the enzyme within a binder matrix and numerous other forms of immobilizing the isomerases have been suggested.
It is also known that isomerases are less susceptible to deactivation when the isomerization reaction is conducted in the presence of one or more metal ion activators. The metal ion activators and the requirements will depend primarily upon the specific type of isomerase. When an isomerase is isolated from a new source or in a different form, it is conventional to establish its metal ion activator requirements..sup.1 Suppliers of commercial isomerases customarily provide technical information with respect to its metal ion activator requirements in a glucose isomerization process. FNT 1 - References cited herein are illustrative of the establishment of metal ion activator requirements in an isomerization process.
In the commercial production of enzymatically isomerized glucose syrups, the metal ion activator concentration is carefully controlled at a prescribed level. Excessive or insufficient metal ion activator concentrations are avoided. For the prolonged time intervals necessary to complete the isomerization reaction, an excessive amount of metal ion activator frequently represses isomerase efficacy (e.g., isomerase poisoning), can present health hazards (e.g., toxic), may impart undesirable flavor or color to isomerized glucose syrup as well as adversely affecting syrup stability, which necessitate additional refining or filtration processing steps to remove these impurities therefrom (e.g., ion exchange treatment). If the metal activator ion concentration is too low, stabilization against deactivation will not be achieved. Thus, it is customary for the isomerized glucose syrup producer to maintain the metal ion activator concentrations at the lowest possible level yet sufficiently high to preserve the isomerase's activity.
Certain isomerases require a sole metal ion activator whereas others evince greater stability when co-metal ion activators are present in the isomerization media. When co-metal ion activators have a stabilizing effect upon the isomerase, normally one metal ion activator will be essential while the other co-metal ion activator will not significantly increase the isomerase activity unless the required metal ion activator is also cooperatively present in the reaction media.
It has been reported that certain isomerases are activated by conducting the isomerization reaction in the presence of thiol activating reagents such as glutathione and cysteine (e.g., see J. Agri. Chem. Soc. Japan 36, No. 12, 1013-1016, 1962, by Y. Takasaki et al.; J. Biol. Chem. 218, 535, 1956 by M. J. Palleroni et al.; J. Am. Chem. Soc. 77, 1663, 1955 by M. W. Slein; and Agr. Biol. Chem. Vol. 28, No. 8, pp. 510-516, 1964 by M. Natake et al.). Many of the researchers have also reported that isomerase activity can be markedly decreased by conducting the isomerization reaction in the presence of sulfhydryl binding agents which react with the sulfhydryl group (e.g., cuprous ions such as cuprous sulphate or chloride, p-chloromercuribenzoate, monoidoacetate, mercurous ions, zinc sulphate, etc.). Conducting the isomerization reactions in the presence of oxidizing agents, including nascent oxygen, have also been reported as having an inhibitory effect upon isomerase activity.